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. 2024 Jan 12;15(1):2302246. doi: 10.1080/21655979.2024.2302246

Figure 5.

Figure shows domain architecture of proteins with 2, 3, 4, 6, or 9 copies of a CD40-specific nanobody (VHH) and their CD40 stimulating activity with the 6 and 9 copies containing versions being highly active. Data showing purification and lack of aggregation of the variants with 4, 6, and 9 nanobody domains are also included.

Oligomeric sdAb:cd40 variants display strong CD40 agonism. (a) Domain architecture of oligomeric sdAb:CD40 variants. DANA refer to the D256A-N297A mutation preventing/reducing FcγR binding of IgG1. (b) Western blot analysis of reduced sdAb:cd40 variants. Position of MW (kDa) markers are indicated. Asterixes refer to not fully reduced protein species. (c) U2OS cells were stimulated overnight with the different VHH:CD40 variants and finally IL8 production was determined by ELISA. (D,E) the indicated constructs were purified by affinity chromatography and analyzed by SDS-PAGE (D) and gel filtration (E). Dotted arrows indicate flag peptide remained from the affinity purification.